Natural products have a proven record of providing a significant fraction, either directly or as lead compounds, of human medicines. Among natural products, the terpenoids (isoprenoids) stand out as being the largest class (>50,000 already known), with the 20-carbon diterpenoids targeted here forming a significant fraction of these (>12,000 known). The extensive diversification of diterpenoids indicates that the manifold hydrocarbon skeletons that can be formed from this C20 backbone provide privileged scaffolds for derivation of biological activity. Indeed, a number of diterpenoids are used as pharmaceuticals (e.g., the anti-cancer paclitaxel/TaxolTM and antibiotic mutilins) or research tools (e.g., the protein kinase C activating and tumor promoting phorbol esters, and adenylate cyclase activator forskolin). In addition, we have contributed to the discovery that the human pathogen Mycobacterium tuberculosis utilizes diterpenoid metabolism in construction of an immune-modulatory factor. The enzymes that produce the underlying hydrocarbon scaffolds catalyze complex electrophilic reactions that form new carbon-carbon bonds, which are of significant mechanistic interest. Our long-term goal is to engineer enzymes and metabolic pathways for the production of targeted libraries and specific individual terpenoid ?natural? products for pharmaceutical investigation and use. This revised renewal proposal focuses on the critically important scaffold assembling diterpene synthases/cyclases. Here we propose to build on our findings from the previous grant period, which includes discovery of means by which these enzymes may be re-engineered for novel product outcome. In particular, the objective of this proposal is to build on our on-going detailed studies of enzymatic structure-function relationships, and to also demonstrate our understanding of their catalytic mechanism by re-engineering activity, specifically to incorporate the addition of water to generate hydroxylated products. Such re-engineering will be enabled by the innovative approach taken here of combining quantum chemical calculations on simplified theozymes to identify key functional groups and their optimal positioning relative to high-energy intermediates, which will guide Rosetta based enzyme (re)design, followed by biochemical characterization of the (re)designed enzymes, with the results then informing subsequent (re)design efforts. We further will investigate the potential pharmaceutical activity of the resulting hydroxylated diterpenes, leveraging our modular metabolic engineering system used to characterize biochemical function. In particular, the resulting recombinant bacteria will be fed to a variety of Caenorhabditis elegans disease models to determine the ability of the the produced hydroxylated diterpenes to alleviate the relevant symptoms.